Embedding biocatalysts in a redox polymer enhances the performance of dye-sensitized photocathodes in bias-free photoelectrochemical water splitting

Dye-sensitized photoelectrodes consisting of photosensitizers and molecular catalysts with tunable structures and adjustable energy levels are attractive for low-cost and eco-friendly solar-assisted synthesis of energy rich products. Despite these advantages, dye-sensitized NiO photocathodes suffer from severe electron-hole recombination and facile molecule detachment, limiting photocurrent and stability in photoelectrochemical water-splitting devices. In this work, we develop an efficient and robust biohybrid dye-sensitized NiO photocathode, in which the intermolecular charge transfer is enhanced by a redox polymer. Owing to efficient assisted electron transfer from the dye to the catalyst, the biohybrid NiO photocathode showed a satisfactory photocurrent of 141±17 μA·cm−2 at neutral pH at 0 V versus reversible hydrogen electrode and a stable continuous output within 5 h. This photocathode is capable of driving overall water splitting in combination with a bismuth vanadate photoanode, showing distinguished solar-to-hydrogen efficiency among all reported water-splitting devices based on dye-sensitized photocathodes. These findings demonstrate the opportunity of building green biohybrid systems for artificial synthesis of solar fuels.

2) P. 4, line 149.The authors comment on a large photocurrent density of 181 μA/cm2, but from Fig. 3b it is clear that the measured current does not reach zero upon removal of the light input.The photocurrent must be determined as the difference between the current measured under light subtracted by the value under dark (dark current).Thus, the photocurrent value provided should be adjusted to a slightly lower value in order to account for this evidence.
3) P. 4 and after.The manuscript would benefit from inclusion of addifional characterizafion data related to the novel photocathode.Parficularly, photoacfion spectra should be measured and IPCE data provided.From these data, internal quantum efficiency (IQE or APCE) can be then extracted by taking into account the absorpfion of the photoelectrode.Finally, knowing the injecfion yield and the APCE, the charge collecfion efficiency can be esfimated.The resulfing value can be highly relevant to discuss about the improved charge transport arising from the presence of the polymer redox-relay.4) P. 6, line 191.The transient absorpfion studies are well conducted and speak in favor of the acfive role of the polymer matrix in enhancing charge transport.I am wondering, however, whether the authors consider measuring the transient absorpfion in the presence of the hydrogenase in order to follow the electron transfer from the reduced polymer to the catalyst and thus confirm that this process "is not limifing the device's photcurrent".

5)
Partly connected to the previous point.I am wondering why the authors limit their analysis as a funcfion of the pH to a very narrow range (6-7.5).Is it related to electrode/catalyst instability?What about shifting to acidic pH where the HER should become easier?
Reviewer #2 (Remarks to the Author): The manuscript reports an original biohybrid strategy for overall solar-driven water splifting in photoelectrochemical cells.It focuses on the development of an efficient dye-sensifized photocathode based on a [FeFe]-hydrogenase as proton reducfion catalyst.The integrafion of the redox-acfive polymer PolyV in the electrode architecture proved to be key to this strategy; it resulted in an excepfional increase in performance as well as stability, enabling to record unprecedented STH efficiencies for a dyesensifized photocathode in a bias-free tandem cell configurafion.Kudos to the authors for this very nice piece of work!This thorough study, based on carefully performed and clearly described experiments, is of broad and general interest to the community and clearly deserves to be published in Nature Communicafions.Minor comments and quesfions are outlined below.
-References related to the preparafion of the nanoporous NiO films are missing (page 10, line 322).Did the authors use a specific procedure to increase the size of the pores in order to facilitate the penetrafion of the polymer and the enzyme (which looks opfimal from the SEM measurements), as well as the diffusion of the buffer electrolyte throughout the electrode structure?-It is stated page 10, line 327 that the opfimized PolyV concentrafion is 1.5 mg.L-1, which is not so clear on Figure 3a… The plateau is rather reached at a concentrafion of 3 mg.L-1… -In Figure 1, the blue arrow depicfing the electron transfer from NiO to the sensifizer is upside down.
-Page 11, line 384: « The Faradaic efficiency for H2 and O2 was calculated with equafion (1).The amount of enzyme in this calculafion was based on how much enzyme was deposited on the electrode.» ?? The Faradaic efficiency does not depend on the amount of catalyst present at the surface of the electrode.

Reviewer #3 (Remarks to the Author):
This work represents a real breakthrough in the field, with the design of a water-splifting device based on an innovafive dye-sensifized photocathode with a biocatalyst displaying unprecedented performance.This significant result is the fruit of the collaborafion of three groups sharing their respecfive experfise and innovafive findings accumulated over the last few years.The experiments are well performed and the data well analyzed.The manuscript is very clear and is adapted to the wide readership of the journal.I would recommend the publicafion of this manuscript after my unique, important comment is addressed.
Page 5, lines 164-70: How the remaining HER acfivity of the enzyme after the 5 hours is measured?Regarding the same experiments, how do the authors explain that the photocurrent remains confinuous (Fig. 3c) while 22 % of the enzymes are no longer acfive?How do the authors explain the evolufion of the faradaic yield in Fig. 3d during the first hour?To ensure that this is not a latent fime to generate an acfive iron-based catalyst, the authors could introduce a non-acfive iron complex (modificafion of the dithiolate ligand) into the acfive site of the hydrogenase and verify that the system is not acfive or reconsfitute the enzyme with the FeS clusters (without the acfive site).Page 4, line 136: conc PolyV, a plateau at 3 mg.mL-1 in the figure and 2 mg.mL-1 in the text Dear reviewers, Thank you very much for reviewing our manuscript of " Embedding biocatalysts in a redox polymer enhances the performance of Dye-Sensitized Photocathodes in Biasfree Photoelectrochemical Water Splitting" (NCOMMS-23-60841-T).All comments received are very helpful for us to improve the manuscript.

Response to Reviewer 1
Reviewer's comment: The manuscript by Tian and co-workers describes the preparation of a photocathode for hydrogen evolution based on sensitized NiO and incorporation of a hydrogenase in a redox-active viologen-based polymer.The photocathode is highly active towards photoelectrochemical hydrogen production reaching unprecedented photocurrent densities.Interestingly, the photocathode has been also combined with a BiVO4 photoanode to perform bias-free photoelectrochemical water splitting.The manuscript is interesting and the results unprecedented in the field so that it may warrant publication.The following comments should be, however, addressed before the manuscript can be accepted.1. P. 3, line 93.The authors describe the design motif associated with the photocathode and point out the requirement of no direct contact between the catalyst and the FTO substrate.
However, this statement is in sharp contrast with the results obtained by SEM-EDS where a rather homogeneous distribution of Fe is apparent (Fig. 2).Please clarify this issue.

Response:
We thank the reviewer for the comment.We don't think the hydrogenase can cross the NiO blocking layer as the blocking layer is very compact and prepared from a sputtering method.As the blocking layer is very thin and also the hydrogenase might fall to the cross-section when we cut the film for the SEM test, it is hard to clearly see the boundary between NiO blocking layer and hydrogenase.
2. P. 4, line 149.The authors comment on a large photocurrent density of 181 μA/cm 2 , but from Fig. 3b it is clear that the measured current does not reach zero upon removal of the light input.
The photocurrent must be determined as the difference between the current measured under light subtracted by the value under dark (dark current).Thus, the photocurrent value provided should be adjusted to a slightly lower value in order to account for this evidence.

Response:
We thank the reviewer for raising this important consideration.After the subtraction by the dark current, the photocurrent stated in the revised manuscript has been changed to 14117 A•cm -2 , which is still among the best photocurrent obtained from dyesensitized NiO photocathode for fuel production.The corresponding changes are made on Page 1, 2 and 4.
3. P. 4 and after.The manuscript would benefit from inclusion of additional characterization data related to the novel photocathode.Particularly, photoaction spectra should be measured and IPCE data provided.From these data, internal quantum efficiency (IQE or APCE) can be then extracted by taking into account the absorption of the photoelectrode.Finally, knowing the injection yield and the APCE, the charge collection efficiency can be estimated.The resulting value can be highly relevant to discuss about the improved charge transport arising from the presence of the polymer redox-relay.

Response:
We appreciate the suggestion.We have measured the photocurrent of the photocathode under LED lamps with wavelength at 470 nm, 590 nm and 630 nm (Supplementary Fig. 9).The IPCE and APCE data is provided for each sample with or without PolyV, showing the assisted charge transport by the redox polymer.4. P. 6, line 191.The transient absorption studies are well conducted and speak in favor of the active role of the polymer matrix in enhancing charge transport.I am wondering, however, whether the authors consider measuring the transient absorption in the presence of the hydrogenase in order to follow the electron transfer from the reduced polymer to the catalyst and thus confirm that this process "is not limiting the device's photcurrent".
Response: Thank the reviewer for this suggestion.In our previous work, we have demonstrated the fast intermolecular electron transfer between the redox polymer and the hydrogenase by cyclic voltammetry (Nat. Catal. 2021, 4, 251-258).In addition, the pH independency of the photocurrent (Supplementary Fig. 10) also indicates that the electron transfer between the polymer and the enzyme is not the limiting step in the overall electron transfer pathway.In the fs-TA experiment, the reduced polymer was observed; however, the signal was weak, posing challenges for conducting subsequent TA experiments in the presence of hydrogenase.Therefore, in this work we are only able to monitor the electron extraction from the dye to PolyV. 5. Partly connected to the previous point.I am wondering why the authors limit their analysis as a function of the pH to a very narrow range (6-7.5).Is it related to electrode/catalyst instability?What about shifting to acidic pH where the HER should become easier?
Response: We agree with the reviewer that the environment with acidic pH is preferable for HER.In this work, the catalysis experiments were conducted in a neutral pH range (6-7.5),due to the best stability and activity of the enzyme in a neutral environment.It is possible to obtain high HER currents at pH<6, but at the expense of a limited stability.